Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisor: Jeanne H. Freeland-Graves. Vita. Includes bibliographical references.

No description available.

Lee Tracy Yuen Han. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 168-187). / Abstracts in English and Chinese. / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vi / Table of contents --- p.viii / List of tables --- p.xii / List of figures --- p.xiii / List of abbreviations --- p.xiv / Chapter Chapter 1: --- Prenatal diagnosis --- p.1 / Chapter 1.1 --- Historical overview --- p.1 / Chapter 1.1.1 --- Prenatal diagnosis --- p.1 / Chapter 1.1.2 --- Circulating fetal nucleated cells --- p.2 / Chapter 1.1.3 --- Cell-free fetal DNA in maternal plasma --- p.3 / Chapter 1.2 --- Biological characteristics of circulating DNA --- p.4 / Chapter 1.3 --- Origin of circulating DNA --- p.6 / Chapter 1.4 --- Clinical applications of circulating fetal DNA --- p.7 / Chapter 1.5 --- Quantitative aberrations in circulating fetal DNA --- p.9 / Chapter 1.6 --- Epigenetic approach in detecting circulating fetal DNA --- p.11 / Chapter Chapter 2: --- Epigenetics --- p.14 / Chapter 2.1 --- Historical overview --- p.14 / Chapter 2.2 --- Mechanisms of DNA methylation --- p.15 / Chapter 2.3 --- Roles of DNA methylation --- p.17 / Chapter 2.4 --- Aberrations in DNA methylation --- p.20 / Chapter 2.5 --- Epigenetic diagnostic markers --- p.22 / Chapter 2.6 --- Significance of epigenetic markers in noninvasive prenatal diagnosis --- p.23 / Chapter 2.7 --- Aim of thesis --- p.23 / Chapter Chapter 3: --- Materials and methods --- p.25 / Chapter 3.1 --- Preparation of samples --- p.25 / Chapter 3.1.1 --- Collection of placental tissues --- p.25 / Chapter 3.1.2 --- Preparation of plasma and blood cells --- p.25 / Chapter 3.2 --- Nucleic acid extraction --- p.26 / Chapter 3.2.1 --- DNA extraction from placental tissues --- p.26 / Chapter 3.2.2 --- DNA extraction from plasma --- p.26 / Chapter 3.2.3 --- DNA extraction from blood cells --- p.29 / Chapter 3.3 --- Bisulfite genomic sequencing --- p.30 / Chapter 3.3.1 --- Principles of bisulfite modification --- p.30 / Chapter 3.3.2 --- Primer design for bisulfite sequencing --- p.31 / Chapter 3.3.3 --- Bisulfite genomic sequencing --- p.31 / Chapter 3.3.3.1 --- Bisulfite modification --- p.31 / Chapter 3.3.3.2 --- Bisulfite genomic sequencing --- p.32 / Chapter 3.3.4 --- Data and statistical analysis --- p.38 / Chapter 3.4 --- MALDI-TOF Mass Spectrometry (MS) --- p.39 / Chapter 3.4.1 --- Principle of MALDI-TOF MS and MassEXTEND assay --- p.39 / Chapter 3.4.2 --- Methylation-sensitive restriction enzyme digestion and MassEXTEND assay for PDE9A and H19 --- p.41 / Chapter 3.5 --- Quantitative measurements of nucleic acids --- p.44 / Chapter 3.5.1 --- Principles of real-time quantitative PCR --- p.44 / Chapter 3.5.2 --- Methylation-specific qMSP assay for M- and U-PDE9A --- p.47 / Chapter Chapter 4: --- Systematic screening of 8 CGIs in third trimester pregnancy --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.2 --- Methods --- p.50 / Chapter 4.2.1 --- Subjects --- p.50 / Chapter 4.2.2 --- Experimental design --- p.51 / Chapter 4.3 --- Results --- p.54 / Chapter 4.3.1 --- Methylation profile of 8 CGIs in maternal blood cells and paired placental tissues --- p.54 / Chapter 4.3.1.1 --- Methylation profile of PDE9A in third trimester pregnancy --- p.56 / Chapter 4.3.1.2 --- Methylation profile of PPP1R2P2 in third trimester pregnancy --- p.60 / Chapter 4.3.1.3 --- Methylation profile of LOC115376 in third trimester pregnancy --- p.65 / Chapter 4.3.1.4 --- Methylation profile of AM L1 in third trimester pregnancy --- p.71 / Chapter 4.3.1.5 --- Methylation profile of COL6A2 in third trimester pregnancy --- p.78 / Chapter 4.3.1.6 --- Methylation profile of PRDM15 in third trimester pregnancy --- p.82 / Chapter 4.3.1.7 --- Methylation profile of CG1111 in third trimester pregnancy --- p.86 / Chapter 4.3.1.8 --- Methylation profile of CGI121 in third trimester pregnancy --- p.90 / Chapter 4.3.2 --- Methylation profile of regions upstream and downstream of PDE9A in maternal blood cells and paired placental tissues --- p.94 / Chapter 4.3.2.1 --- Methylation profiles of PDE9A_A and PDE9ÁؤB in third trimester pregnancy --- p.96 / Chapter 4.3.2.2 --- Methylation profile of PDE9ÁؤC in third trimester pregnancy --- p.100 / Chapter 4.4 --- Discussion --- p.104 / Chapter Chapter 5: --- "Methylation analysis of PPP1R2P2, PDE9A, PDE9A B and PDE9ÁؤC in first trimester pregnancy" --- p.108 / Chapter 5.1 --- Introduction --- p.108 / Chapter 5.2 --- Methods --- p.109 / Chapter 5.2.1 --- Subjects --- p.109 / Chapter 5.2.2 --- Experimental design --- p.109 / Chapter 5.3 --- Results --- p.110 / Chapter 5.3.1 --- Methylation profile of PPP1R2P2.Region A in first trimester pregnancy. --- p.110 / Chapter 5.3.2 --- Methylation profile of PDE9A in first trimester pregnancy --- p.115 / Chapter 5.3.3 --- Methylation profile of PDE9A B in first trimester pregnancy --- p.119 / Chapter 5.3.4 --- Methylation profile of PDE9A C in first trimester pregnancy --- p.123 / Chapter 5.4 --- Discussion --- p.127 / Chapter Chapter 6: --- "Methylation analysis of PPP1R2P2, PDE9A and PDE9ÁؤB in Trisomy 21 pregnancy" --- p.129 / Chapter 6.1 --- Introduction --- p.129 / Chapter 6.2 --- Methods --- p.130 / Chapter 6.2.1 --- Subjects --- p.130 / Chapter 6.2.2 --- Experimental design --- p.131 / Chapter 6.3 --- Results --- p.131 / Chapter 6.3.1 --- Methylation profile of PPP1R2P2 in trisomy 21 placental tissues --- p.131 / Chapter 6.3.2 --- Methylation profile of PDE9A in trisomy 21 placental tissues --- p.136 / Chapter 6.3.3 --- Methylation profile of PDE9A B in trisomy 21 placental tissues --- p.140 / Chapter 6.4 --- Discussion --- p.144 / Chapter Chapter 7: --- Investigation of imprinting status of PDE9A --- p.145 / Chapter 7.1 --- Introduction --- p.145 / Chapter 7.2 --- Methods --- p.147 / Chapter 7.2.1 --- Subjects --- p.147 / Chapter 7.2.2 --- Experimental design --- p.147 / Chapter 7.3 --- Results --- p.149 / Chapter 7.3.1 --- SNP detection in enzyme digested placental tissues by H19 assay --- p.149 / Chapter 7.3.2 --- SNP detection in enzyme digested placental tissues by PDE9A assay --- p.151 / Chapter 7.4 --- Discussion --- p.153 / Chapter Chapter 8: --- Detection of U-PDE9A DNA sequences in maternal plasma --- p.155 / Chapter 8.1 --- Introduction --- p.155 / Chapter 8.2 --- Methods --- p.156 / Chapter 8.2.1 --- Subjects --- p.156 / Chapter 8.2.2 --- Experimental design --- p.156 / Chapter 8.3 --- Results --- p.157 / Chapter 8.3.1 --- Detection of U-PDE9A DNA sequences in maternal plasma --- p.157 / Chapter 8.3.2 --- Clearance of U-PDE9A DNA sequences from maternal plasma after delivery --- p.157 / Chapter 8.4 --- Discussion --- p.160 / Chapter Chapter 9: --- Conclusion and future perspectives --- p.162 / Chapter 9.1 --- Methylation profiles of CpG islands on chromosome 21q --- p.162 / Chapter 9.2 --- Investigation of imprinting status of PDE9A --- p.164 / Chapter 9.3 --- Development of a universal epigenetic marker --- p.165 / Chapter 9.4 --- Future perspectives --- p.166 / References --- p.168

The discovery of fetal-derived circulating nucleic acids in maternal plasma has opened up new opportunities for non-invasive prenatal diagnosis. This non-invasive means of obtaining fetal genetic materials is safer than invasive tissue-sampling procedures, which are associated with a small but finite chance of fetal loss. Over the past decade, the detection of fetal DNA in maternal plasma has evolved from dependency on discriminative genetic markers, such as Y-chromosome-specific loci or paternally-inherited polymorphisms, to detection of circulating RNA, fetal-specific methylation or by massively parallel sequencing. Fetal-specific methylation, or fetal epigenetic marker, does not require prior knowledge of the sex or polymorphic status of the fetus and thus can be applied in essentially all pergnancies. This thesis focuses on the development of this kind of marker for non-invasive monitoring and detection of pre-eclampsia and fetal aneuploidies. / The first part of this thesis describes the use of a reported fetal epigenetic marker, RASISF1A, to measure the fetal DNA concentrations in maternal plasma of pre-eclamptic subjects versus gestational-age-matched controls. The second part of this thesis describes a systematic search for potential epigenetic markers for pre-eclampsia and the second commonest fetal aneuploidy, trisomy 18. Numerous approaches for methylation profiling are described, such as methylation-specific polymerase chain reaction (MSP), bisulfite sequencing, a mass spectrometry-based platform (the Epityper assay), and methylated DNA immunoprecipitation coupled with tiling array analysis (MeDIP-chip). Using MeDIP-chip, I selected the most promising fetal epigenetic markers on chromosome 18, and further characterised their detection in maternal plasma. The final part of this thesis describes an approach called epigenetic-genetic (EGG) chromosome dosage for the detection of trisomy 18 based on those markers. I have demonstrated that it is feasible to detect fetal trisomy 18 by analysing maternal plasma in as early as the first trimester. / This thesis illustrates different strategies for methylation profiling and presented two examples of applying DNA methylation for the non-invasive prenatal assessment of pregnancy-associated disorders and fetal chromosomal aneuploidies. I envision that a similar strategy could be developed for other pregnancy-related diseases to broaden the application of epigenetic markers in non-invasive prenatal diagnosis. / Tsui, Wai Yi. / Adviser: Y.M. Dennis Lo. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 198-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Prenatal diagnosis is an established obstetrics practice in many countries. Currently available methods to obtain fetal materials for a definitive diagnosis involve invasive procedures such as chorionic villus sampling and amniocentesis. Due to the invasive nature of the procedures, confirmatory testing is usually recommended only for pregnant women who are screened as being high risk of bearing a fetus with abnormalities. There has been an urge to develop safer alternatives in obtaining fetal genetic materials for prenatal assessment. Thus, the discovery of circulating fetal DNA in maternal plasma and serum has opened up new possibilities for noninvasive prenatal diagnosis. / The use of genetic markers such as Y chromosomal sequences from a male fetus or paternally-inherited polymorphic markers has been well-described in the literature. However, there is an obvious limitation on the use of these markers because they are gender- and/or polymorphism-dependent. This thesis focuses on the development of a universal fetal marker, namely SERPINB5 (encoding maspin), based on the intrinsic epigenetic differences between the placenta (the major contributor of fetal genetic materials in maternal plasma) and maternal blood cells (postulated to be the predominant source of cell-free DNA in the circulation). Analysis of the methylation profile of the SERPINB5 promoter in the placenta and maternal blood cells, and the development of methods and protocols to detect the differentially methylated SERPINB5 promoter molecules are described. The application of this epigenetic marker in prenatal assessment of the fetal chromosomal aneuploidy is illustrated by an epigenetic allelic ratio (EAR) approach. Basically, the ratio of a single-nucleotide polymorphism (SNP) on the placenta-derived SERPINB5 molecules is shown to be deviated in the maternal plasma from trisomic pregnancies when compared to the euploid ones. Results described in this thesis show the promising potential for the EAR approach for the noninvasive prenatal detection of fetal chromosomal aneuploidies. In addition, a novel approach for methylation analyses on the amplification and detection of restriction enzyme-digested DNA fragments will be discussed. This thesis has essentially described the evolution of a fetal epigenetic marker from basic science to potential clinical application. / Tong, Yu Kwan. / Adviser: Yuk-Ming Dennis Lo. / Source: Dissertation Abstracts International, Volume: 69-02, Section: B, page: 0953. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 149-172). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / School code: 1307.

Kan, Hoi Lam. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 103-124). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENTS --- p.vi / TABLE OF CONTENTS --- p.viii / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiii / LIST OF ABBREVIATIONS --- p.xiv / PUBLICATION --- p.xvi / Chapter SECTION I: --- BACKGROUND --- p.1 / Chapter Chapter 1: --- Hepatocellular Carcinoma (HCC) --- p.2 / Chapter 1.1. --- Epidemiology of HCC --- p.3 / Chapter 1.2. --- Etiology of HCC --- p.3 / Chapter 1.2.1. --- Cirrhosis --- p.4 / Chapter 1.2.2. --- Hepatitis virus --- p.4 / Chapter 1.2.3. --- Plant carcinogens --- p.5 / Chapter 1.2.4. --- Miscellaneous factors --- p.6 / Chapter 1.3. --- Clinical presentation of HCC --- p.6 / Chapter 1.4. --- Existing diagnostic tests for HCC --- p.6 / Chapter 1.4.1. --- Alpha-fetoprotein (AFP) --- p.7 / Chapter 1.4.2. --- Imaging --- p.7 / Chapter 1.5. --- Treatment of HCC --- p.8 / Chapter 1.5.1. --- Surgical Resection and Transplantation --- p.8 / Chapter 1.5.2. --- Tumor Ablation or Embolization --- p.8 / Chapter 1.5.3. --- Chemotherapy and Radiotherapy --- p.9 / Chapter 1.6. --- Tumor marker development for HCC detection --- p.10 / Chapter 1.6.1. --- Oncofetal antigens and glycoprotein antigens --- p.11 / Chapter 1.6.2. --- Enzymes and isoenzymes --- p.12 / Chapter 1.6.3. --- Growth factors --- p.12 / Chapter 1.6.4. --- Genetics and epigenetics - mRNA and methylation --- p.13 / Chapter Chapter 2: --- Hypermethylation of tumor suppressor genes in cancer --- p.14 / Chapter 2.1. --- Cancer epigenetics --- p.14 / Chapter 2.2. --- DNA methylation in normal cells --- p.15 / Chapter 2.3. --- Physiological role of DNA methylation in normal cells --- p.18 / Chapter 2.4. --- Aberrant DNA methylation in cancer --- p.19 / Chapter 2.4.1. --- DNA hypomethylation in cancer --- p.20 / Chapter 2.4.2. --- DNA hypermethylation in cancer --- p.20 / Chapter 2.5. --- Development of methylation markers in tumor diagnosis --- p.21 / Chapter 2.5.1. --- Methods for the analysis of DNA methylation markers --- p.22 / Chapter 2.5.2. --- Detection of tumor-associated methylated DNA in the circulation of cancer patients / Chapter 2.6. --- Aim of thesis --- p.27 / Chapter SECTION II: --- MATERIALS AND METHODS --- p.28 / Chapter Chapter 3: --- Methods for detecting DNA methylation --- p.29 / Chapter 3.1. --- Subject recruitment --- p.29 / Chapter 3.2. --- Sample collection and processing --- p.29 / Chapter 3.2.1. --- Tumor tissue samples --- p.29 / Chapter 3.2.2. --- Peripheral blood samples --- p.29 / Chapter 3.3. --- DNA extraction --- p.30 / Chapter 3.3.1. --- Plasma samples --- p.30 / Chapter 3.3.2. --- Blood cells --- p.33 / Chapter 3.3.3. --- Tumor tissue --- p.33 / Chapter 3.4. --- Quantitative analysis of methylated DNA using methylation-sensitive restriction enzyme-mediated real-time quantitative PCR (MSRE-qPCR) --- p.34 / Chapter 3.4.1. --- Methylation-sensitive restriction enzyme-mediated real-time quantitative PCR --- p.34 / Chapter 3.4.3. --- Real-time PCR primer design --- p.36 / Chapter 3.4.4. --- Duplex real-time PCR --- p.40 / Chapter 3.4.5. --- "Real-time detection of GSTP1, SOCS1, A PC, pl6 and ACTB sequences" --- p.41 / Chapter 3.4.6. --- Statistical analysis of real-time PCR results --- p.41 / Chapter 3.5. --- "Methylation study of GSTP1, SOCS1, APC, pl6 and ACTB in tumor tissues and blood cells using bisulfite sequencing" --- p.46 / Chapter 3.5.1. --- Principle of bisulfite modification --- p.46 / Chapter 3.5.2. --- Bisulfite conversion --- p.47 / Chapter 3.5.3. --- Sequencing primer design --- p.47 / Chapter 3.5.4. --- Conventional PCR after bisulfite treatment --- p.49 / Chapter 3.5.5. --- Cloning and bisulfite genomic sequencing --- p.53 / Chapter 3.5.6. --- Data acquisition and interpretation --- p.54 / Chapter SECTION III: --- DEVELOPMENT OF METHYLATION MARKERS IN HCC DETECTION / Chapter Chapter 4: --- Evaluation of the real-time PCR assay for quantification of methylated tumor suppressor genes --- p.57 / Chapter 4.1. --- Development of real-time PCR assays --- p.57 / Chapter 4.2. --- Methylation analyses by bisulfite sequencing were concordant with the real-time quantification results --- p.61 / Chapter Chapter 5: --- Clinical application of methylated markers in the detection of hepatocellular carcinoma --- p.69 / Chapter 5.1. --- Demographics of HCC patients and HB V carriers --- p.69 / Chapter 5.2. --- Quantitative analysis of hypermethylated tumor suppressor genes in tumor and plasma samples --- p.71 / Chapter 5.3. --- Effect of cirrhosis on the plasma methylated tumor suppressor gene concentrations --- p.77 / Chapter 5.4. --- Changes in the concentration of the tumor suppressor genes one month after surgical resection of the cancer --- p.81 / Chapter 5.5. --- Concurrent use of serum AFP level and plasma methylated markers for HCC diagnosis --- p.84 / Chapter 5.6. --- Prognostic value of plasma methylated TSGs --- p.86 / Chapter SECTION IV: --- DISCUSSION --- p.90 / Chapter Chapter 6: --- Discussion --- p.91 / Chapter 6.1. --- Tumor and plasma detection of hypermethylated tumor suppressor genes --- p.92 / Chapter 6.2. --- No effect of cirrhosis on plasma methylated DNA level --- p.94 / Chapter 6.3. --- Clearance of methylated TSG sequences after tumor resection --- p.95 / Chapter 6.4. --- Concurrent use of serum AFP level and the presence of methylated markers in the plasma in HCC diagnosis --- p.95 / Chapter 6.5. --- Prognostic significance of circulating methylated tumor markers --- p.96 / Chapter SECTION V: --- CONCLUDING REMARKS --- p.98 / Chapter Chapter 7: --- Conclusions and future perspectives --- p.99 / REFERENCES --- p.103

Thesis (PhDAgric)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Maize is undoubtedly South Africa's most important field crop. The identification of markers and genes for traits of interest is important to sustain the improvement of maize cultivation. Northern corn leaf blight (NClB) is a disease that occurs worldwide and can dramatically reduce yield. A number of single dominant resistance genes have been identified for NClB and some have been mapped. Currently there are no simple PCR markers for any of these resistance genes, making markerassisted selection (MAS) difficult. The aim of this study was to develop PCR markers for the NClB resistance genes Ht1, Ht2, Ht3 and Htn1 in maize. To accomplish this, the AFlP (amplified fragment length polymorphism) technique was first optimised. The results indicated that the Mlul/Msel restriction enzyme combination produces a higher percentage of polymorph isms when compared to the PstllMsel enzyme combination. It was also shown that the enzyme combination plays an important role in the percentage of polymorphic fragments observed, whereas the number of restriction enzymes used in AFlP analysis only significantly affects the total number of fragments scored. Populations segregating for the different resistance genes were not available for this study. Nearly-isogenic lines (Nils) were used in combination with AFlP technology to identify markers that map close to the genes. AFlP markers common in at least two resistant or susceptible lines were cloned and converted to PCR markers. Two commercially available recombinant inbred line (Ril) populations were then used to map the identified markers. For Htn1 fifteen polymorphic fragments were present in both resistant lines. They were selected for sequence specific marker conversion. Seven of the fifteen sequence characterized amplified region (SCAR) markers were polymorphic on the Nil pairs and five mapped to one region of maize chromosome 8.05/06. Twenty-one AFlP markers were identified for Ht1 and four SCAR markers were polymorphic In the Ht1 Nils. Three of these were mapped to chromosome 2.07. Three AFlP markers were identified for Ht2 of which two were converted to SCAR markers. Both SCAR markers were polymorphic on the Ht2 Nils and mapped to chromosome 8.05/06. On the Ht3 NILs, four AFLP markers were identified and two converted SCAR markers and one microsatellite marker (bnlg1666) were polymorphic. One of the SCAR markers and the microsatellite marker were mapped to chromosome 7.04 using a RIL population. This reports the first tentative mapping position for the Ht3 locus. The next step was to determine if a set of marker alleles could be used in a number of Htn 1 resistance lines to identify a common donor region selected by the breeders. Nine markers consisting of five SCAR markers, three converted RFLP markers and one microsatellite marker were used on 16 Htn1 resistant lines. The marker allele of us3 was in 12 of the 16 lines in coupling with Htn1 resistance. Second was the marker us5 in 11 of the 16 lines. Using this data 14 of the 16 lines shared a common introgressed region between the markers us3 and us5. A further common introgressed region between 11 of the inbred lines was found between the markers us14 and asg17. The last aim of this study was to propose a new marker technique that might be more successful than the AFLP technique in the identification of markers closely linked to genes. A new marker approach was identified where a MITE (Hbr) primer was used as an anchor primer in combination with resistance gene analog primers. This was found to be a highly polymorphic marker technique that could be used to identify markers and possibly candidate genes. It is a robust technique, which is affordable since amplifications occur from undigested genomic DNA and the primers mainly amplify fragments from genic regions. / AFRIKAANSE OPSOMMING: Mielies (Zea mays) is ongetwyfeld Suid Afrika se belangrikste lanbou gewas. Vir volgehoue opbrengs verbetering is die identifisering van merkers en gene vir belangrike eienskappe noodsaaklik. Noordelike blaarskroei (NBS) kan opbrengs wesenlik kan beïnvloed. Tans is daar reeds "n aantal enkel weerstandsgene geïdentifiseer, maar geen PKR-merkers is beskikbaar vir merker gebaseerde seleksie nie. Die doelwit van hierdie studie was om PKR-merkers te ontwikkel vir vier enkel weerstands gene (Ht1, Ht2, Ht3 en Htn1) teen NBS in mielies. Om die doelstelling te bereik is die AFLP-tegniek eers geoptimiseer. Op grond van waargenome aantal polimorfismes, was Mlul/Mse/"n beter restriksie ensiem kombinasie as Pstl/Msel. In die studie is ook bewys dat die aantal (meer as twee) restriksie ensieme wat gebruik word slegs die aantal fragmente, en nie die persentasie polimorfismes, wesenlik beïnvloed nie. Geen segregerende populasie was vir die verskillende gene beskikbaar nie. Naby isogeniese lyne (NILe) is daarom in kombinasie met die AFLP-tegniek gebruik om merkers te identifiseer wat naby die gene karteer. Alleenlik polimorfiese merkers wat in ten minste twee weerstand biedende of vatbare lyne voorgekom het, is gekloneer en omgeskakel na PKR-merkers. Daarna is twee kommersiële rekombinante ingeteelde lyn populasies gebruik om die gene te karteer. Vyftien fragmente is gevind wat gekoppel was met die Htn1 weerstand. Sewe van hierdie merkers is omgeskakel in polimorfiese SCAR-merkers waarvan vyf gekarteer is in een gebied op chromosoom 8.05/06. Een-en-twintig AFLP-merkers is geïndentifiseer vir Ht1 en vier is omgeskakel na polimorfiese SCAR-merkers. Drie hiervan is gekarteer op chromosoom 2.07. Drie AFLP-merkers is geïndetifiseer vir Ht2 waarvan 2 omgeskakel is na polimorfiese SCAR-merkers. Altwee hierdie merkers is gekarteer op chromosoom 8.05/06. Op die Ht3 lyne is vier AFLP-merkers geïdentifiseer waarvan twee omgeskakel is na polimorfiese SCAR-merkers. Een mikrosatelliet merker (bnlg1666) is ook gevind wat die selfde polimorfiese patroon wys op die Ht3 lyne. Die mikrosateliet en een van die SCAR-merkers het gekarteer op chromosomale posisie 7.04. Hierdie is die eerste tentatiewe posisie vir die Ht3 lokus. Die volgende stap was om te bepaal of "n stel polimorfiese merker-allele gebruik kan word om die donor DNA-segment te identifiseer wat die plantteiers geselekteer het. Nege PKR-merkers wat bestaan het uit vyf SCAR-merkers, 3 omgeskakelde RFLP merkers en een mikrosateliet is gebruik op 16 Hnt1 weerstandslyne. Us3 was die merker alleel wat in die meeste gevalle gekoppel was met die Htn1 weerstandslyne (12/16). Tweede was die merker us5 (in 11 van die 16 lyne). Uit die data blyk dit dat 14 van die 16 lyne "n donor segment het wat beide merkers us3 en us5 bevat. Merkers us14 en asg17 het in 11 van die 16 bestande lyne saam voorgekom. Die laaste doelstelling van hierdie studie was om "n nuwe tegniek te ontwikkel wat dalk meer suksesvol as AFLPs kan wees om merkers te identifiseer nabyaan gene. "n Nuwe tegniek word voorgestel waar "n MITE (Hbr) inleier gebruik kan word in kombinasie met weerstandgeen-analoog inleiers. Dit is gevind dat hierdie kombinasie van inleiers "n hoogs polimorfiese band patroon gee en dat die merkers ook dalk kandidaat-gene kan wees. Die tegniek is maklik uitvoerbaar, relatief goedkoop en maak gebruik van onverteerde genomiese DNA. Die fragmente wat geamplifiseer word is hoofsaaklik vanaf geenryke areas.

Cheung Siu-king. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves [103-113]). / Abstracts in English and Chinese. / ACKNOWLEDGMENT --- p.i / TABLE OF CONTENTS --- p.ii / LIST OF ABBREVIATIONS --- p.vi / LIST OF TABLES --- p.vi / LIST OF FIGURES --- p.ix / ABSTRACT (ENGLISH VERSION) --- p.x / ABSTRACT (CHINESE VERSION) --- p.xii / Chapter 1. --- INTRODUCTION --- p.1 / Chapter 1.1. --- ADOLESCENT IDIOPATHIC SCOLIOSIS --- p.1 / Chapter 1.1.1. --- prevalence and geographic patterns of ais --- p.1 / Chapter 1.1.2. --- CLINICAL ASPECTS OF AIS --- p.3 / Chapter 1.1.3. --- ETIOLOGY OF AIS --- p.8 / Chapter 1.2. --- OBJECTIVES OF THIS STUDY --- p.24 / Chapter 2. --- SUBJECTS AND METHODS --- p.25 / Chapter 2.1. --- STUDY DESIGN --- p.25 / Chapter 2.2. --- SUBJECTS RECRUITMENT --- p.25 / Chapter 2.2.1. --- ais subjects --- p.25 / Chapter 2.2.2. --- control subjects --- p.25 / Chapter 2.2.3. --- GROUPING ACCORDING TO THE CHRONOLOGICAL AGE --- p.26 / Chapter 2.2.4. --- informed Consent --- p.26 / Chapter 2.2.5. --- EVALUATION OF COBB'S ANGLE --- p.26 / Chapter 2.3. --- ANTHROPOMETRIC ASSESSMENTS --- p.26 / Chapter 2.4. --- BMD MEASUREMENTS --- p.28 / Chapter 2.4.1. --- measured by dexa --- p.28 / Chapter 2.4.2. --- measured by pqct --- p.30 / Chapter 2.5. --- BONE FORMATION MARKER ： BALP --- p.32 / Chapter 2.5.1. --- SERUM COLLECTION --- p.32 / Chapter 2.5.2. --- ABBOTT METHODS FOR SERUM ALP ACTIVITY --- p.32 / Chapter 2.6. --- BONE RESORPTION MARKER ： DPD --- p.34 / Chapter 2.6.1. --- PYRILINK-D KITS REAGENT --- p.34 / Chapter 2.6.2. --- CREATININE ASSAY --- p.34 / Chapter 2.7. --- GENETIC MARKER - POLYMORPHISM OF ESTROGEN RECEPTOR GENE --- p.38 / Chapter 2.7.1. --- DIGESTION OF PERIPHERAL BLOOD CELLS --- p.38 / Chapter 2.7.2. --- QUANTITATION OF DNA --- p.39 / Chapter 2.7.3. --- CONFIRMATION OF INTEGRITY OF DNA --- p.39 / Chapter 2.7.4. --- POLYMERASE CHAIN REACTION (PCR) --- p.39 / Chapter 2.7.5. --- REACTION BUFFER --- p.39 / Chapter 2.8. --- STATISTICS --- p.45 / Chapter 3. --- RESULTS --- p.46 / Chapter 3 .1 --- SUBJECT DISTRIBUTION OF AIS AND NORMAL CONTROL --- p.46 / Chapter 3.1.1. --- "mean ages of menarche, breast development and pubic hair development" --- p.47 / Chapter 3.1.2. --- "PUBERTAL STATUES OF DIFFERENT AGE GROUPS EVALUATED BY MENARCHE, BREAST DEVELOPMENT AND PUBIC HAIR DEVELOPMENT" --- p.48 / Chapter 3.2. --- ANTHROPOMETRIC ASSESSMENTS --- p.49 / Chapter 3.2.1. --- OVERALL REVIEW OF ANTHROPOMETRIC ASSESSMENTS --- p.49 / Chapter 3.2.2. --- ANTHROPOMETRIC ASSESSMENTS ACCORDING TO THE CHRONOLOGICAL AGE --- p.50 / Chapter 3.3. --- BMD PROFILE OF AIS PATIENTS --- p.51 / Chapter 3.3.1. --- ABMD MEASURED BY DEXA (OVERALL REVIEW) --- p.51 / Chapter 3.3.2. --- ABMD IN DIFFERENT AGE GROUPS --- p.52 / Chapter 3.3.3. --- VBMD MEASURED BY PQCT (OVERALL REVIEW) --- p.52 / Chapter 3.3.4. --- VBMD IN DIFFERENT AGE GROUPS --- p.53 / Chapter 3.3.5. --- PREVALENCE OF OSTEOPENIA IN AIS PATIENTS --- p.53 / Chapter 3.3.6. --- SYMMETRY OF BILATERAL PROXIMAL FEMUR AND DISTAL TIBIA … --- p.54 / Chapter 3.3.7. --- CORRELATION OF ABMD AND VBMD WITH ANTHROPOMETRIC PARAMETERS AND SPINAL DEFORMITY --- p.54 / Chapter 3.4. --- BONE FORMATION MARKER- BALP --- p.55 / Chapter 3.5. --- BONE RESORPTION MARKER -DPD --- p.56 / Chapter 3.6. --- GENETIC MARKER -ESTROGEN RECEPTOR GENE --- p.57 / Chapter 4 --- DISCUSSION…… --- p.84 / Chapter 4.1 --- BONE MINERAL DENSITY OF AIS PATIENTS --- p.84 / Chapter 4.2 --- ANTHROPOMETRIC MEASUREMENTS --- p.89 / Chapter 4.3 --- BONE BIOCHEMICAL TURNOVER MARKER --- p.91 / Chapter 4.4 --- GENETIC MARKER - ER GENE --- p.97 / Chapter 4.4.1 --- OSTEOPORTIC CANDIDATE GENE- ER GENE --- p.98 / Chapter 4.4.2 --- NO CORRELATION BETWEEN ER GENE AND AIS --- p.99 / Chapter 4.5 --- SUMMARY --- p.100 / Chapter 5. --- CONCLUSION --- p.101 / BIBLIOGRAPHY --- p.XIV / APPENDIX --- p.XXV

No description available.

Colorectal cancer (CRC) is the fourth most common malignancy in the developed Western countries and represents the second leading cause of death from cancer after lung cancer. Despite better diagnostic tools and improvement of surgical standards, the prognosis of this disease is still unsatisfactory with a mean 5-year cancer-specific survival of approximately 50%, mainly due to our inability to predict liver failure secondary to hepatic involvement and still poorly effective palliative treatments. The metastatic cascade is a complex, multistep process driven by progressive accumulation of genetic alterations which result in proto-oncogene activation and inactivation of tumour suppressor genes. Among the additional pathways involved in this process, secretion of angiogenesis factors, proteolysis of the extra-cellular matrix (ECM) molecules, and probably inhibition of apoptosis are also known to facilitate tumour progression. We undertook a retrospective study in a series of paraffin-embedded human colonic tissues to investigate the prognostic significance of new tumour markers as predictors of liver metastasis and survival in sporadic CRC. This research was conducted in three parts: first, immunohistochemical studies of protein markers and development of a new quantitative method of measurement in a subset of the immunostained sections using a colour video image analysis (VIA) procedure; second, concomitant determination of the expression of the bcl-2 gene at the messenger RNA (mRNA) level, using a more specific methodology of in situ hybridisation (ISH) and investigation of the relationship between bcl-2 mRNA and its encoded protein expression; and third, investigation of microsatellite alterations at four loci using a fluorescent microsatelllite polymerase chain reaction (PCR) assay coupled with an automated DNA sequencer. In our initial immunohistochemical experiment, we found a good correlation between colour VIA and semiquantitative evaluation of nm23 immunoreactivity (IR) confirming the validity of such quantitative analysis (Pearson's correlation coefficient r=0.88; P<0.001). Furthermore, overexpression of nm23 was associated with an increased risk of developing liver metastases (logrank test for trend, P<0.001) and cancer-related death (P=0.002). We used the same quantitative method to determine the expression of urokinase-type plasminogen activator (u-PA) and c-erbB-2 (HER2/neu) proteins and found that neither predicted patient outcome. However, CRC showing overexpression of u-PA (above 85 pixels) had an increased risk of liver metastasis (P=0.013). Since this was a post hoc analysis we can not be confident that this represents a real effect. There were significant positive correlations among expression of all three markers, u-PA, c-erbB-2 and nm23 proteins (u-PA vs c-erbB-2, Spearman rank correlation coefficient, P=0.003; u-PA vs nm23, P<0.001; c-erbB-2 vs nm23, P=0.001) suggesting that, in vivo, all proteins interact or are similarly regulated. Semiquantitative analysis of the vascular endothelial growth factor (VEGF) protein showed that expression of VEGF was significantly reduced in the metastatic liver tumours compared to their matched primary ones (Wilcoxon test, P=0.002), suggesting VEGF activity to be secondarily down-regulated once the tumour cells reach the hepatic parenchyma. There was no strong evidence from our data that the level of VEGF in the primary tumour could predict risk of liver metastasis or survival duration. Finally, when semiquantitatively assessing five protein markers (nm23, p53, c-erbB- 2, u-PA, and VEGF) individually or in combination, we found that only nm23 protein expression was positively related to the risk of liver metastasis (logrank test, P<0.001); p53 protein expression was only marginally associated (P=0.091). Furthermore, patients with Dukes' stage B tumours showing positive expression of nm23 protein also demonstrated an increased risk of liver metastasis (P=0.001). Although the risk of developing liver secondaries was statistically correlated with the number of positive markers (NPM) and the cumulative intensity score (CIS) (logrank test for trend, P=0.004 and P=0.001 respectively), these two parameters did not improve the predictive value over and above that of nm23 protein alone. These results suggest that the only marker of the five we studied that provides prognostic information about the risk of developing liver metastasis in CRC is nm23. Its evaluation may be particularly useful in selecting high risk Dukes' stage B patients who should be considered for adjuvant chemotherapy. In the second part of this study, immunohistochemical analysis using a monoclonal mouse antibody to the bcl-2 protein and in situ hybridisation using digoxigenin-labelled bcl-2 cRNA probes were carried out on the same paraffin-embedded specimens and semiquantitatively assessed. These specimens also included adenomas with various degrees of dysplasia. The expression of bcl-2 protein was gradually and significantly lost during the progression from moderately dysplastic adenoma to primary CRC [moderate/severe dysplasia: Mann-Whitney U-test, P=0.0001; severe dysplasia/primary CRC: P=0.027], whereas the cellular expression of bcl-2 mRNA was progressively increased during the dysplasia/adenoma-carcinoma neoplastic sequence. Our observation suggests that in a proportion of CRCs the bcl-2 proto-oncogene expression may be down-regulated at a post-transcriptional level. In the final section of this study, we investigated the possible relationship between loss of heterozygosity (LOH) and microsatellite instability (MIN) at four microsatellite loci spanning the 17q21-23 region that includes the nm23-H1 and nm23- H2 genes, to the risk of liver metastasis and nm23 protein expression. Genomic DNA was extracted from the same series of paraffin-embedded colorectal specimens and a fluorescent PCR coupled with DNA fragment analysis in an automated DNA sequencer was applied. In 45% and 48% of the primary and secondary lesions, LOH was present in at least one locus. We found a positive correlation when looking for a trend comparing the fraction of sites with LOH at these loci to risk of liver recurrence (logrank test for trend, P=0.005). This remained an independent predictor after adjusting to T-stage (Multivariate Cox regression, P=0.022), N-stage (P=0.007), or Dukes' stage (P=0.012). MIN was present in 2 loci in 41% and 30% of the primary and secondary tumours respectively. Considering only the Dukes' B tumours, we found that an increasing number of sites showing MIN was associated with a reduced risk of liver recurrence (logrank test for trend, P=0.032). When comparing LOH and MIN status of the primary and matched liver secondary tumours with their corresponding normal tissue samples, we found concordant genomic alterations in 72% (NME1 locus) to 43% (D17S579). Finally, we observed a trend in association between the proportion of loci with LOH and nm23 positivity ( ?? 2 test for trend, P=0.024). These findings suggest that, genomic alterations in the 17q21-23 region may affect prognosis of CRC as well as regulation of the nm23 protein expression via a stillmechanism.